Dynamics and Regulations of BimEL Ser65 and Thr112 Phosphorylation in Porcine Granulosa Cells during Follicular Atresia
Dynamics and Regulations of BimEL Ser65 and Thr112 Phosphorylation in Porcine Granulosa Cells during Follicular Atresia
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猪卵泡闭锁过程中猪颗粒细胞 BimEL Ser65 和 Thr112 磷酸化的动态和调控
DOI：
10.3390/cells9020402
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发表时间：
2020-02-01
期刊：
影响因子：
6
通讯作者：
Zeng, Shenming
中科院分区：
文献类型：
--
作者：
Yang, Feng;Chen, Yanhong;Zeng, Shenming
BimEL蛋白通过调节颗粒细胞凋亡参与滤泡闭锁，但目前对BimEL磷酸化在滤泡闭锁过程中的动态变化了解甚少。本研究旨在探讨BimEL关键磷酸化位点的变化及其上游调控途径。首先，采用Western blotting检测不同处理后健康(H)、轻度闭锁（SA）和闭锁(A)卵泡颗粒细胞（GC）和培养的GC中BimEL-Ser65和BimEL-Thr112磷酸化水平（p-BimEL-S65, p-BimEL-T112）。接下来，研究BIM对应位点突变对GC细胞凋亡的影响。最后，用激酶抑制剂研究了两个磷酸化位点的通路。结果表明，H组GC中p- bmel - s65含量高于SA和A组，而p- bmel - t112含量相反。促生存因子FSH、IGF-1上调p- bmel - s65水平，促凋亡因子热应激上调p- bmel - t112水平。与野生BimEL过表达相比，GC过表达的BimEL- s65a（用Ala代替Ser65）突变体的凋亡率显著高于野生BimEL，而过表达的BimEL- t112a细胞的凋亡率无显著差异。此外，通过特异性抑制剂抑制ERK1/2或JNK通路可降低p-BimEL-S65和p-BimEL-T112的水平。综上所述，p- bmel - s65和p- bmel - t112水平在卵泡闭锁期间逆转。促生存因子通过ERK1/2促进p- bmel - s65水平抑制GC细胞凋亡，而促凋亡因子通过JNK上调p- bmel - t112水平诱导GC细胞凋亡。
BimEL protein is involved in follicular atresia by regulating granulosa cell apoptosis, but the dynamic changes of BimEL phosphorylation during follicular atresia are poorly understood. The aim of this study was to explore the changes of key BimEL phosphorylation sites and their upstream regulatory pathways. First, the levels of BimEL-Ser65 and BimEL-Thr112 phosphorylation (p-BimEL-S65, p-BimEL-T112) in granulosa cells (GC) from healthy (H), slightly-atretic (SA), and atretic (A) follicles and in cultured GC after different treatments were detected by Western blotting. Next, the effects of the corresponding site mutations of BIM on apoptosis of GC were investigated. Finally, the pathways of two phosphorylation sites were investigated by kinase inhibitors. The results revealed that p-BimEL-S65 levels were higher in GC from H than SA and A, whereas p-BimEL-T112 was reversed. The prosurvival factors like FSH and IGF-1 upregulated the level of p-BimEL-S65, while the proapoptotic factor, heat stress, increased the level of p-BimEL-T112 in cultured GC. Compared with the overexpression of wild BimEL, the apoptotic rate of the GC overexpressed BimEL-S65A (replace Ser65 with Ala) mutant was significantly higher, but the apoptotic rate of the cells overexpressing BimEL-T112A did not differ. In addition, inhibition of the ERK1/2 or JNK pathway by specific inhibitors reduced the levels of p-BimEL-S65 and p-BimEL-T112. In conclusion, the levels of p-BimEL-S65 and p-BimEL-T112 were reversed during follicular atresia. Prosurvival factors promote p-BimEL-S65 levels via ERK1/2 to inhibit GC apoptosis, whereas proapoptotic factor upregulates the level of p-BimEL-T112 via JNK to induce GC apoptosis.